Project Summary/Abstract This project will validate a novel sample type for diagnosis of active pulmonary tuberculosis (TB). Pulmonary TB is usually detected by analysis of sputum, a viscous material derived from deep in the airways of patients. Diagnosis would be simplified by using a safer, simpler, and more uniform sample. We hypothesize that oral swab analysis (OSA) can meet this need. In OSA an interior surface of the mouth is gently scraped with a disposable swab, which is then tested for Mycobacterium tuberculosis DNA by using quantitative PCR (qPCR). Oral swabs are much easier to collect, handle, and process than sputum. Swabbing is painless, non-invasive, and requires just seconds; an entire classroom or workplace can be sampled in minutes. Patients who are unable to produce sputum can easily be sampled by OSA. Preliminary results demonstrate that M. tuberculosis DNA routinely accumulates in diagnostically useful amounts on surfaces (tongues and interior cheeks) within the mouths of adult and pediatric TB patients. The diagnostic yield of OSA in adults approaches or matches that of sputum. This project will refine, characterize, and validate OSA as an alternative or supplement to sputum analysis for pulmonary TB diagnosis. The following Aims will be completed: 1) To optimize and validate OSA sampling methodologies; 2) to characterize behavioral, microbiological, physiological, and clinical factors affecting OSA results; 3) to quantify the performance of optimized OSA methodologies relative to diagnostic gold standards. OSA could facilitate point-of-care diagnostic strategies, improve diagnostic yields relative to sputum alone, and greatly simplify the care of patients who cannot produce sputum. It could also facilitate active case finding strategies, leading to reduced TB transmission in many settings. Thus, validation of OSA could transform the global fight against TB.